The biofuels industry uses biomass, primarily from plants, to produce soluble sugars that are subsequently fermented to create fuels such as ethanol, butanol, adipate, methylfuran, isoprenes, and biodiesel for human use. The complex structure of biomass, particularly the diversity of cellulosic structures that make up a large portion of plant materials, make the efficient and economical deconstruction of biomass into soluble sugars a difficult challenge for the industry.
Currently, biomass may be chemically pretreated to facilitate the partial decomposition of biomass structure. Specifically, chemical treatment allows for more complete contact of enzymes or microbes to the biomass structure. Chemicals used for such treatments may include acids, steam, ionic liquids, alkaline hydrogen peroxide, or high pressure liquid ammonia (AFEX).
After any chemical pretreatment, the biomass undergoes enzymatic hydrolysis to produce solubilized sugars suitable for fermentation. Because of the complex structure of biomass, many different enzymes are necessary for complete biomass deconstruction, including cellulases, glycohydrolases, xylanases, and xylobiosidases, mannases and mannosidases, arabinofuranosidases, lichinases, esterases, pectinases, and other enzyme types. Enzymes used in biomass deconstruction exist naturally in bacteria and other organisms, and researchers are currently engaged in extensive enzyme discovery efforts to characterize and isolate previously unknown enzymes that may prove useful in biomass deconstruction.
In nature, many approaches have evolved for the enzymatic deconstruction of cellulose. One class of natural cellulolytic enzymes are freely diffusible enzymes that bind to cellulose only in the sense that an enzyme active site will recognize the substrate and bind to a specific arrangement of chemical bonds in order to perform catalysis, the hydrolytic cleavage of the glycosidic bond.
A second class of natural cellulolytic enzymes bind to cellulose through carbohydrate binding domains, cellulose binding domains, cellulose binding modules, or other binding domains on the enzyme surface. The binding domains facilitate the attachment of the enzyme to the cellulose to effect the deconstruction of cellulose. Such enzymes are not attached to the cell, and must exist outside of the cell to have function.
A third class of natural cellulolytic enzymes also interact with cellulose to effect its deconstruction, but are additionally bound to a bacterial cell wall. Such enzymes are found on the outer surface of bacterial cells.
A fourth class of natural cellulolytic enzymes include cellulolytic, hemicellulolytic, pectinolytic, and/or esterolytic enzymes that are assembled into multiprotein complexes called cellulosomes, which are complexes of enzymes created by bacteria such as Clostridium and Bacteroides. Cellulosomes assemble and function outside of the bacterial cells that create the component enzymes. The cellulosomal enzymes are attached to a large, multimodular, noncatalytic subunit called scaffoldin. Scaffoldin has domains known as cohesins, which interact with other domains called dockerins. Cohesins integrate dockerin-tagged enzymes into the cellulosome complex. Scaffoldin and some cellulosomal enzymes also contain carbohydrate binding domains, cellulose binding domains, cellulose binding modules, or other binding domains which bind to cellulose, hemicellulose, starch, pectin, chitin or other polysaccharide structures.
Cellulosome architecture is the consequence of the types and specificities of the interacting cohesin and dockerin domains, borne by the different cellulosomal subunits (Haimovitz et al., 2008, Proteomics 8: 968-979), and is further affected by the presence of carbohydrate binding domains. It has been shown that it is possible to create designer chimeric cellulosomes through the modification of cohesin and dockerin domains (Fierobe et al., 2005, J. Biol. Chem. 280: 16325-16334). It has also been shown that artificial scaffoldin proteins can be created to accomplish the function of the scaffoldin while not relying on the domain structure or order of the natural scaffoldin to achieve this function.
As illustrated by the great diversity cellulolytic enzymes, many combinations of enzymes and proteins are involved in natural cellulose deconstruction. Further evidence of the great complexity and diversity of possible cellulose degradation pathways is provided by the genomic sequencing of microbes and fungi, and by bioinformatic analysis of the metagenomic sequences isolated from all organisms present in a natural environment. For example, recent whole genome sequencing studies of Streptomyces sp. ActE isolated from the Sirex wood wasp revealed 127 separate genes that are plausibly involved in the breakdown of carbohydrates (C. Currie, et al., Streptomyces sp. ACTE, whole genome shotgun sequencing project, NCBI. Reference Sequence: NZ_ADFD00000000.1). In another recent study assaying gene expression during growth on cellulose in C. thermocellum ATCC 27405 using controlled growth rate microarrays, 348 of the organism's 3191 genes were expressed, and 34 of the expressed genes had uncharacterized export signals (Riederer, Takasuka, Makino, Stevenson, Bukhman, Fox, unpublished work).
The complexity of biomass deconstruction as a biological problem makes conventional single enzyme assays inadequate for devising new and more efficient methods needed to develop a sustainable and economical biofuels industry. Although many new organisms containing useful enzymes may be discovered and the resulting genomes may be sequenced, the successful selection of the most promising new organisms for such purposes is difficult at best, and effective tools are not currently available to effectively focus proteomics efforts using any newly discovered gene sequences. Furthermore, conventional single enzyme studies do not adequately address the complexity of the biological problem. Thus, there is a need in the art for methods to efficiently and quickly discover effective combinations of enzymes and/or coordinated enzyme complexes for use in facilitating biomass transformation.